TEL-AML1 Transgenic Zebrafish Model of Human Leukemia

描述(申请人提供)：近期目标：建立TEL-AML1转基因斑马鱼模型，并研究加重白血病发生的继发性遗传事件。事业发展目标：在白血病研究领域发展独立的事业。研究项目：t(12；21)染色体易位产生TEL-AML1融合基因，是儿童癌症中最常见的结构遗传异常，仅与前体B细胞急性淋巴细胞白血病(ALL)相关。有证据表明，TEL-AML1易位通常发生在胎儿造血过程中的子宫，并构成了一种启动或首发突变，这对白血病的发生是必要的，但还不够。 这项建议的目的是确定TEL-AML1诱导的白血病发生所需的继发性遗传事件。为了验证TEL-AML1融合蛋白需要继发遗传事件才能诱导白血病转化的中心假说，有两个特定的目标：1)建立稳定表达TEL-AML1融合蛋白的转基因斑马鱼系，并评估转基因表达对斑马鱼的造血和白血病发生的影响；2)通过研究正常斑马鱼TEL-AML1融合蛋白和组蛋白乙酰转移酶活性的变化，建立转TEL-AML1融合蛋白斑马鱼“二次打击”模型；同时，在转基因斑马鱼中启动逆转录病毒插入突变筛选，目的是确定在所有发育过程中与TEL-AML1融合蛋白协同作用的癌症基因。实现这些目标的可用资源包括我们已建立的TEL-AML1转基因创始人及其后代、斑马鱼组蛋白乙酰转移酶突变体和表达斑马鱼特异报告基因的逆转录病毒。 斑马鱼系统特别适合于这些实验，因为它提供了一个具有多样性的生物学、遗传灵活性、保守的造血发育以及进行转基因和后续突变的机会的脊椎动物模型。在染色体融合蛋白存在的情况下，执行大规模表型驱动的突变筛查以识别加速白血病发展的协同事件的能力代表了该模型的独特优势。在这些研究中识别白血病途径中的关键基因可能会识别白血病特异性治疗的新靶点。 虽然这份申请是为了K22职业发展奖，但我相信，在国家癌症研究所拥有三年的博士后奖学金为我发展独立的癌症研究生涯做好了准备，对这份申请的积极反应将极大地促进这一过渡。我的长期目标是确定与儿童白血病发展相关的基因事件，而执行拟议研究的能力是朝着这一重要目标迈出的一步。

DESCRIPTION (provided by applicant): Immediate Goal: To generate TEL-AML1 transgenic zebrafish model and investigate the secondary genetic events that accentuate leukemia development. Career Development Goal: To develop an independent career in leukemia research. Research Project: The t(12;21) chromosomal translocation, which generates the TEL-AML1 fusion gene, is the most frequent structural genetic abnormality in childhood cancer, and is exclusively associated with precursor B-cell acute lymphoblastic leukemia (ALL). Evidence suggests that the TEL-AML1 translocation usually occurs In Utero during fetal hematopoiesis and constitutes an initiating or first-hit mutation that is necessary but insufficient for leukemia development. The aim of this proposal is to identify the secondary genetic events required for the development of TEL-AML1-induced leukemia. The two specific aims designed to test the central hypothesis that TEL-AML1 fusion protein requires secondary genetic events to induce leukemic transformation are: 1) To generate stable transgenic zebrafish lines expressing the TEL-AML1 fusion protein, and evaluate the effects of transgene expression on zebrafish hematopoiesis and leukemic development; 2) To develop a "second-hit" model in TEL-AML1 transgenic zebrafish by investigating the altered expression of the normal zebrafish TEL and histone acetyltransferase activity, both implicated in TEL-AML1 leukemic development, and in parallel, by initiating a retroviral insertional mutagenesis screen in transgenic zebrafish aimed at identifying cancer genes that synergize with the TEL-AML1 fusion protein in ALL development. Resources available to carry out these aims include our established TEL-AML1 transgenic founders and their progeny, a zebrafish mutant for histone acetyltransferase, and a retrovirus expressing zebrafish-specific reporter gene. The zebrafish system is uniquely suitable for these experiments in that it presents a vertebrate model with a versatile biology, genetic flexibility, conserved hematopoietic development, and the opportunity to conduct transgenesis and subsequent mutagenesis. The ability to perform a large-scale phenotype-driven mutational screen to identify cooperating events that accelerate leukemic development in the presence of chromosomal fusion proteins represents the unique advantage of this model. The identification of crucial genes in the leukemic pathway in these studies may identify novel targets for leukemia specific therapies. Although this application is for a K22 career development award, without an institutional affiliation, I believe that having a postdoctoral fellowship at the National Cancer Institute for three years prepared me to develop an independent cancer research career, and that the favorable response to this application would significantly facilitate this transition. My long-term goal is to identify the genetic events associated with the development of leukemia in children, and the ability to perform the proposed studies is one step towards this vital goal.